Fluorescence suppression in Raman spectroscopy ... - OSA Publishing

Fluorescence suppression in Raman spectroscopy using a time-gated CMOS SPAD Juha Kostamovaara,1,* Jussi Tenhunen,2 Martin Kögler,2 Ilkka Nissinen,1 Jan Nissinen,1 and Pekka Keränen1 1

University of Oulu, Department of Electrical Engineering, Electronics Laboratory, FIN-90014 Oulu, Finland 2 VTT Electronics, 90570 Oulu, Finland * [email protected]

Abstract: A Raman spectrometer technique is described that aims at suppressing the fluorescence background typical of Raman spectra. The sample is excited with a high power (65W), short (300ps) laser pulse and the time position of each of the Raman scattered photons with respect to the excitation is measured with a CMOS SPAD detector and an accurate timeto-digital converter at each spectral point. It is shown by means of measurements performed on an olive oil sample that the fluorescence background can be greatly suppressed if the sample response is recorded only for photons coinciding with the laser pulse. A further correction in the residual fluorescence baseline can be achieved using the measured fluorescence tails at each of the spectral points. ©2013 Optical Society of America OCIS codes: (120.0120) Instrumentation, measurement, and metrology; (120.6200) Spectrometers and spectroscopic instrumentation; (300.6450) Spectroscopy, Raman.

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#197491 - $15.00 USD Received 17 Sep 2013; revised 21 Nov 2013; accepted 6 Dec 2013; published 13 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.031632 | OPTICS EXPRESS 31632

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1. Introduction Raman spectroscopy is based on measurement of the intensity distribution of inelastically scattered photons from a monochromatic light source as a function of wavelength. Usually a CW (continuous wave) laser operating in the visible, near-infrared or near-ultraviolet range is used as a light source for illuminating the sample [1,2]. The laser light interacts with molecular vibrations and rotations in the sample system with the result that the energy (wavelength) of the scattered photons is shifted up or down (anti-Stokes or Stokes scattering). For a certain vibrational or rotational mode of a specific molecule, the change in the energy of the Raman scattered photon (the “Raman shift”) is independent of the excitation photon energy. The Raman shift is typically expressed in wavenumbers [1/cm], i.e. as the difference between the excitation laser and the Raman emission wavenumbers. The Raman spectrum, i.e. the spectral distribution of the intensity of the Raman scattered photons, is unique for each chemical component and thus provides selectivity for a specific chemical component. Since the spectral distribution of the Raman scattering is a concentration-weighted linear combination of the Raman spectra of the pure components, the chemical composition of the sample can be calculated from the measured spectrum using chemometric calibration models [1]. Furthermore, dangerous components or counterfeit products can be identified in the sample. Since absorption phenomena are weak in the VIS and NIR (visible and near-infrared) spectral regions where Raman spectroscopy is typically applied, Raman spectroscopy provides for easy and practical sampling, as does NIR spectroscopy, but where NIR spectroscopy yields information only on functional groups containing hydrogen (CH, OH, NH,..), the Raman spectrum provides a full set of vibrational and rotational spectral information, as typically obtainable only by combining the MIR and FIR (mid-infrared, farinfrared) spectroscopic techniques. On the other hand, Raman avoids the sampling difficulties entailed in these latter techniques, which result from the high absorption coefficients of the materials and the optical elements themselves within the wavelength regions concerned. For fibre-optic sampling, the spectral transmission of the fibre materials is even more suitable for Raman than for NIR spectroscopy, while in the case of MIR and FIR spectroscopies the technology for fibre-optic sampling is very limited and expensive [2]. Being the result of a scattering process, unlike NIR, MIR and FIR spectra, the Raman spectrum as measured through container walls, blisters, plastic bags, etc. provides information only on the enclosed material and not on the container, provided that the excitation and pick-up beams do not overlap at the container but only in the material that the package contains. This allows the contents to be studied without destroying or opening the packages [3]. Furthermore, Raman spectroscopy gives better resolved spectral lines, and since it has a low sensitivity for water, it is also better applicable to the study of biological and biochemical samples, which typically have a high water content [4]. #197491 - $15.00 USD Received 17 Sep 2013; revised 21 Nov 2013; accepted 6 Dec 2013; published 13 Dec 2013 (C) 2013 OSA 16 December 2013 | Vol. 21, No. 25 | DOI:10.1364/OE.21.031632 | OPTICS EXPRESS 31633

Unfortunately, the measured Raman spectrum is typically masked by a strong fluorescence background in most potential applications. This is due to the fact that the probability of Raman scattering (cross-section) is much lower than that of fluorescence [1,2,5]. A strong fluorescence background gives rise to two problems. Firstly, it becomes the dominant element in the photon shot noise and thus detracts from the SNR (signal-to-noise ratio), and secondly, even if the Raman bands are narrow and the fluorescence has quite a smooth, featureless spectrum, errors in the mathematical estimation and removal (background subtraction) of the fluorescence increase with increasing fluorescence levels and result in increasing errors in both material identification and concentration measurement applications. As a result, in spite of the obvious advantages of Raman spectroscopy, the strong fluorescence background has so far restricted its use in most otherwise potential applications in the agricultural, food and oil industries, security control and crime investigations, for example. Due to this limitation, Raman spectroscopy is nowadays generally used for the analysis of samples that do not absorb the excitation laser, “white powders” and synthetic, and thus clean, substances, e.g. in the pharmaceuticals industry. An established laboratory practice for coping with the fluorescence problem given the existing instrumentation is “photobleaching”, in which the specimen to be analysed is illuminated with the excitation laser over an extended period of time, typically several minutes, in order to destroy the fluorescent chromophores [6]. The method is nevertheless slow, it works only with some materials, it may induce chemical or physical changes in the samples and it certainly cannot be applied to living organisms. Fluorescence has been effectively suppressed up to now by using near-infrared laser excitation, e.g. NdYAG 1064 nm, instead of the traditional shorter laser excitation wavelengths (400 nm – 830 nm), but this requires the use of InGaAs or Ge detectors, which produce dark current and read noise levels that are several orders of magnitude higher than the Si CCD which is predominantly used with traditional, shorter wavelength excitation (400 nm – 830 nm). This results in an increased noise level and a need for high excitation laser power (>1000 mW vs. typically a few tens to a few hundred mW with CCD-based systems) and long integration times (30 min to several hours vs. from a few seconds to a few minutes with CCD-based systems) [1]. Fortunately, since Raman scattering has a lifetime of much less than a picosecond, whereas fluorescence lifetimes are typically in the range of few thousand picoseconds or even tens of nanoseconds, it is possible to suppress the fluorescence background to a great extent if short, intensive laser pulses are used to illuminate the sample rather than CW radiation and the sample response is recorded only during these short pulses, see Fig. 1 and [5,7–9], for example, and references therein. Thus, by “time-gating” the measurement to the period of the laser pulse, the total intensity of the fluorescence in the recorded spectrum can be substantially reduced. This results in an improved signal-to-noise ratio and/or shorter measurement time, since the photon noise of the recorded fluorescence emission, which is proportional to the square root of the fluorescence baseline intensity, is now markedly reduced. Furthermore, the accuracy of the baseline of the Raman spectrum is improved, which also leads to greater accuracy in both material identification and quantitative analysis applications. Several Raman spectrometer measurement set-ups that employ the above time-gating principle have been presented. For example, a high-speed optical shutter based on a Kerr cell is used to realize the time-gate function in [5] and [9], while a photo multiplier and a time gate fulfil the same purpose in [7]. The measurement of a time-gated Raman spectrum with a combination of a mode-locked laser, spectrograph and ICCD (intensified CCD) to achieve a 200ps temporal resolution has also been demonstrated [8]. Although functional in principle and able to demonstrate the potential of the time-gating method, the above systems are either highly sophisticated, physically large and expensive, or capable of measuring only a single wavelength band of the spectrum at a time, so that they require long measurement times and are thus unsuitable for off-laboratory applications.


Fig. 1. Principle of time-gated Raman spectrum measurement.

We present here a measurement set-up which utilizes a time-gated CMOS single photon avalanche detector (SPAD) to determine the time position of the Raman scattered photons with respect to the laser shot. The results show that the width of the time gate can be less than 100ps, which results in a very efficient reduction in the fluorescence background, since this reduction is roughly proportional to the ratio of the time constant of the fluorescence emission to the width of the time gate. Moreover, since CMOS SPADs are realizable in 2D arrays, this method can be used for simultaneous measurement of the whole Raman spectrum by targeting each of the columns of the 2D detector array to a specific Raman wavelength band. Thus the method proposed here could pave the way to the development of a totally new family of Raman spectrometers intended for applications in which suppression of the fluorescence background is important. Similar techniques based on measurement of the time position of photons with CMOS SPAD detectors have been used earlier in fluorescence studies, e.g. in [10]. The time-gating of a CMOS SPAD in Raman measurement was proposed and preliminarily demonstrated in [11], and then also in [12,13] with a CMOS SPAD 2D array having a minimum time-gate of 700ps. A 2D CMOS SPAD array with even shorter time gate was studied in [14]. Compared with [11] and [12,14], we present here a technique for measuring the complete 2D time interval – wavenumber spectrum of the sample, and especially for demonstrating the effectiveness of the sub-ns time-gating in achieving fluorescence suppression. Reference measurements obtained using other Raman spectrometer techniques are also shown. 2. Measurement configuration 2.1 Measurement principle The measurement set-up and instrumentation used are shown in Fig. 2. A pulsed laser source is used to illuminate the sample. In order to maximize the effect of the time-gating and resulting fluorescence reduction, the laser pulse width should be at the limit of the system’s time resolution. High average power is still needed to keep the measurement time reasonable, however, due to the low Raman scattering coefficient, and high spectral purity is also required


for the sake of good spectral resolution. Our measurement system used a SHG (second harmonic) Nd:YAG micro-chip solid-state laser as the transmitter, with a wave length, pulse rate, pulse width and peak power of 532nm, 100kHz, 300ps (FWHM) and 65W, respectively. In the proposed measurement set-up a single photon avalanche photon detector (SPAD) is used to detect the Raman scattered photons. This device, which can be realized in standard CMOS technologies [15–17], is basically a pn junction which is reverse-biased above its breakdown voltage, so that even a single photon entering the depleted region can trigger avalanche breakdown in the device. With a proper bias arrangement, the breakdown can directly produce a high-speed logic-level pulse for the measurement electronics without the need for a sensitive preamplifier. Thus this device is in fact a digital detector in nature (Geiger mode) and its sensitivity is limited by the shot noise of the signal and by the dark counts produced by thermal generation and any tunnelling effects. It should be noted, however, that in order to achieve a linear system response (in this case a Raman spectrum) using a Geiger mode detector, the probability of photon detection in a single laser pulse should be markedly less than one, which is typically the case in a Raman measurement due to the low Raman scattering coefficient. The pulsed laser also triggers a delay generator, which enables the SPAD to operate during and after the laser pulse. Scattered photons are carried through an optical fibre to the spectrograph and to the SPAD-IC. Raman analysis is based on photons counted during a short time-gate window (ΔT in Fig. 1) overlapping with the laser pulse in order to suppress the fluorescence and effective dark count rate of the SPAD detector, the shot noise of which is one of the main noise sources affecting measurement. In this configuration a single CMOS SPAD detector was used so that in order to be able to measure the full Raman spectrum, the detector was moved at the output slit of the spectrometer (Imspector R6E by Specim Inc.) by means of a linear motor stage.

Fig. 2. Block diagram of the time-gated Raman spectrometer.

The SPAD chip was fabricated using 0.35 μm High Voltage CMOS technology and is similar to the structure in [17]. A cross-section of the SPAD chip is shown in Fig. 3. The


diode uses a deep-NWELL cathode and p + anode with a lightly doped p-well guard ring preventing surface breakdown. The SPAD is actively quenched and has a diameter of 20μm for its active area. The shallow depleted high-field region of the detector is quite narrow and thus the photon detection probability is typically only around 20-30%, peaking at about 500nm. On the other hand, due to this narrow depleted region, the time response is good, with a typical jitter of around 100ps (FWHM) [17]. Further details of the SPAD and its performance are given in [11].

Fig. 3. Cross-section of the SPAD.

2.2 Time-gating The schematic and timing diagrams of the SPAD are shown in Fig. 4 (and in Fig. 2). Its operation cycle is controlled via transistors M1 and M2, the operation of which is synchronized with the laser used to illuminate the sample. The bias voltage of the SPAD is kept below the breakdown limit with transistor M1, so that the diode is inoperative between the laser pulses. M1 is then switched off before the laser shot, with the result that the cathode of the SPAD will start to float. The cathode is then biased above the breakdown voltage (VB + VCC > VBR) just before the laser pulse with transistor M2, which is switched ON with a short (